CN105255804B - A kind of catalysis methanol prepares Recombinant organism of isoprene and the preparation method and application thereof - Google Patents
A kind of catalysis methanol prepares Recombinant organism of isoprene and the preparation method and application thereof Download PDFInfo
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- CN105255804B CN105255804B CN201510771229.XA CN201510771229A CN105255804B CN 105255804 B CN105255804 B CN 105255804B CN 201510771229 A CN201510771229 A CN 201510771229A CN 105255804 B CN105255804 B CN 105255804B
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/30—Fuel from waste, e.g. synthetic alcohol or diesel
Abstract
The invention discloses the Recombinant organisms and the preparation method and application thereof that a kind of catalysis methanol prepares isoprene, belong to gene engineering technology field.Recombinant organism provided by the present invention has isoprene route of synthesis, it is overexpressed formate dehydrogenase gene MDH simultaneously, 6 hexulose phosphate synthase gene RmpA, 6 phosphoric acid, 3 ketohexose isomerase gene RmpB and methanol dehydrogenation ras GTPase activating protein ras-GTP Gene A ct.Meanwhile the present invention also provides the methods and application process that build the Recombinant organism.Recombinant organism provided by the present invention can have the features such as transformation efficiency is high, and production cost is low, the conversion ratio of methanol isoprene is up to 21.83% using methanol as substrate biosynthesis isoprene.
Description
Technical field
Prepared the present invention relates to a kind of catalysis methanol Recombinant organism of isoprene and preparation method thereof with
Using belonging to gene engineering technology field.
Background technology
China is world methanol big country, and production capacity is more than the 50% of the world, and domestic production capacity in 2014 is more than 60,000,000 tons, 2015
Year is expected to more than 65,000,000 tons.According to international experience, the rate of capacity utilization be maintained at 81%~82% between be weigh industry production
Can whether superfluous critical point.From the point of view of recent year methanol industry is averaged utilization of capacity data, China's methanol utilization of capacity is always
60%, hereinafter, methanol in 2013 is averaged, the utilization of capacity is 57.13%, in serious superfluous state.Domestic methanol excess capacity,
In addition import methanol prices are relatively low, the price of domestic methanol is caused to continue to decline, maintains within 2014 1500~2000 yuan/ton of left sides
It is right.
The production of methanol fine chemical product can successfully manage the situation of methanol production surplus, for China's methanol work
Important in inhibiting is expanded in the development of industry industry and the in-depth in market.Downstream product than methanol have more development spaces, energy
More arm products are enough produced, and downstream product has more advantage in environmental protection and energy saving aspect compared with methanol, more meets
The new concept of world industry.Based on the above advantage, Downstream Products of Methanol is researched and developed energetically, produces the fine chemical product of high added value
It is the main trend of first alcoholic product development.
The Downstream Products of methanol can be realized by chemical conversion and bioconversion another process.Chemical conversion way
Diameter, it is using methanol as the lower substance of the added values such as waste alkene, dimethyl ether, acetic acid and formaldehyde to study more.Chemistry
Conversion be unable to do without noble metal catalyst, improves production and rises this.Meanwhile the selectivity of catalyst and the conversion ratio of raw material it is relatively low,
The presence for the problems such as by-product is more, seriously polluted seriously constrains the development of industry.In comparison, biological method pollutes
It is small, cost is relatively low, the Efficient Conversion of raw material may be implemented.
The bioconversion of methanol, which utilizes, is in the exploratory stage.Using methanol as carbon source, one can be obtained by microorganism conversion
The higher tunning of a little added values.Eugenio is realized using methanol as carbon source, utilizing works Pichia pastoris synthesis
Hepatitis B virus surface antigen.Pichia pastoris are overexpressed the gene of coding tick worm albumen in Pichia pastoris bodies
Afterwards, methanol-fueled CLC tick worm vaccine can be utilized.Stuart etc. is overexpressed hirudin in Pichia pastoris and synthesizes base into the cell
Cause, fermentation obtain 1.5g/L hirudins in 4 days.Zhang Yuanxing etc. keeps recombinant yeast pichia pastoris thin using the method that methanol glycerol mixed flow adds
Born of the same parents' dry weight reaches 162g/L, finally obtains the hirudin of 1.7g/L；Also the separation of the decoloration of zymotic fluid and hirudin is carried out
It inquires into.Some researchers have carried out the research of methanol single cell protein, find to make raw material using methanol, be sent out by microorganism
The single cell protein of ferment method production contains abundant amino acid, can be as the feed addictive of safety.It is existing about methanol
The research of biological utilisation mainly verifies the production of a few product by the simple culture of Methylotrophic bacterial strain.Total
For, Research foundation is weaker, and there are many urgent problems to be solved.
Isoprene is strategic resource industrial chemicals, unbalanced supply-demand.The 95% of isoprene yield is for synthesizing rubber
Glue, in addition, can also be applied to the fields such as high-quality aviation fuel.It is natural rubber and synthesis to make the primary raw material of tire at present
Rubber (poly- isoprene rubber).Poly- isoprene rubber is to substitute natural rubber because its molecular structure and physico-chemical property are close with natural rubber
Glue manufactures the desirable feedstock of rubber tyre.Increasingly with the fast development of China's automobile industry and China's natural rubber raw material
Shortage is continuously increased the demand of isoprene, it is contemplated that domestic isoprene demand in 2010 will up to 10.54 ten thousand tons,
Rise within 2013 31.04 ten thousand tons, year speedup 46% or so, however, China's isoprene underproduces, need a large amount of imports,
Price is constantly soaring, becomes the important source material for restricting China's related industry development and strategic security.
Currently, industrial mainly prepare isoprene by chemical method from petroleum-based feedstock.Global isoprene master
It produces country and is mainly distributed on European and American areas.Russia is isoprene big producer, and yield accounts for about the whole world 29%,
The U.S. accounts for about 18%, Japan and accounts for about 15%.Secondly it is Brazil, West Europe, Canada etc..The states such as Russia strategically consider, limit
Outlet of the preparing isoprene production technology to China, and China's isoprene natural resources are deficient, the Principles of Chemistry modelling isoamyl two
Alkene production technology workinprocess cost, purity etc. are still immature, restrict the development of related industry, independent research isoprene
Synthetic technology is extremely urgent.As the increasingly depleted of fossil resource and the continuous of price rise, raw material sources are widened, biology is utilized
Technology prepares the hotly contested spot that isoprene has become strategic high-tech in the world.It has been reported that being synthesized for carbon source using glucose
Isoprene, yield and yield are relatively low, while the glucose market price higher than methanol and fluctuates larger.
Invention content
In order to solve the above problem, the present invention provides the bacillus coli gene engineerings that a kind of catalysis methanol prepares isoprene
The preparation method of bacterium, the technical solution taken are as follows：
The purpose of the present invention is to provide the Recombinant organisms that a kind of catalysis methanol prepares isoprene, this is big
Enterobacteria genetic engineering bacterium is to be overexpressed formate dehydrogenase gene MDH, 6- hexulose phosphate synthase gene RmpA, 6- phosphoric acid-
3- ketohexose isomerase gene RmpB and methanol dehydrogenation ras GTPase activating protein ras-GTP Gene A CT.
Preferably, the formate dehydrogenase gene MDH derives from Methylotrophic bacillus (Bacillus
Methanolicus), gene accession number GI：662720579；The 6- hexulose phosphates synthase gene RmpA derives from first
Base auxotype bacillus, gene accession number GI：40074227；6- phosphoric acid -3- ketohexose isomerase gene the RmpB, source
In Methylotrophic bacillus, gene accession number GI：40074228；The methanol dehydrogenation ras GTPase activating protein ras-GTP Gene A CT, source
In Methylotrophic bacillus, gene accession number GI：22654851.
Preferably, the related gene of synthesis isoprene, specially deoxy-D-xylulose sugar 5- phosphate synthase bases are also overexpressed
Because of dxs, deoxy-D-xylulose sugar 5- phosphoric acid reductions enzyme dxr, Isopentenyl diphosphate isomerase idi and isoprenoid synthase gene
Isps。
It is a further object of the present invention to provide a kind of construction method of the Recombinant organism, the steps of this method
It is rapid as follows：
1) it clones or synthesizes and obtain formate dehydrogenase gene MDH, 6- hexulose phosphate synthase gene RmpA, 6- phosphoric acid-
3- ketohexose isomerase gene RmpB and methanol dehydrogenation ras GTPase activating protein ras-GTP Gene A CT, and by gained gene and plasmid pBAD18 enzymes
It cuts back to close, target fragment and carrier；
2) it is connected, and is transferred to using ligase after mixing the carrier obtained by step 1) according to equimolar ratio with target fragment
In competent cell, screening positive clone；
3) incubation step 2) obtained by positive colony, and extract plasmid, four genes described in step 1) be connected to matter
On grain pBAD18, recombinant plasmid pBAD18-MDH-RmpA-RmpB-ACT is obtained；
4) recombinant plasmid of the structure with isoprene route of synthesis, and will be obtained by the recombinant plasmid of gained and step 3)
Recombinant plasmid pBAD18-MDH-RmpA-RmpB-ACT is transformed into together in E. coli BL21 (DE3), is obtained big
Enterobacteria genetic engineering bacterium.
Preferably, step 1) the formate dehydrogenase gene MDH derives from Methylotrophic bacillus (Bacillus
Methanolicus), gene accession number GI：662720579；The 6- hexulose phosphates synthase gene RmpA derives from first
Base auxotype bacillus, gene accession number GI：40074227；6- phosphoric acid -3- ketohexose isomerase gene the RmpB, source
In Methylotrophic bacillus, gene accession number GI：40074228；The methanol dehydrogenation ras GTPase activating protein ras-GTP Gene A CT, source
In Methylotrophic bacillus, gene accession number GI：22654851.
Preferably, the step 2) ligase is T4DNA ligase, Connection Time are 6~12 hours；The competence is thin
Born of the same parents, to pass through heat-shock transformed E.coliDH5 α competent cells；The screening is anti-using coating chloramphenicol or kanamycins
Mild-natured plate is incubated overnight, PCR screenings.
Preferably, the step 4) recombinant plasmid with isoprene route of synthesis, including deoxy-D-xylulose sugar 5- phosphoric acid
Synthase gene dxs, deoxy-D-xylulose sugar 5- phosphoric acid reductions enzyme dxr, Isopentenyl diphosphate isomerase idi and isoprene synthesis
Enzyme gene Isps.
Application of any Recombinant organism in producing isoprene is also in protection scope of the present invention
Within.
The step of application, is as follows：
1) Recombinant organism is activated, seed liquor is obtained；
2) by the seed liquor obtained by step 1), and the culture medium containing kanamycins and chloramphenicol, according to seed liquor：Culture
Base=1~2:It is cultivated to OD after 100~130 ratio inoculation6001.8~2, centrifugation obtains thalline；
3) it is added in fresh culture after being resuspended to the thalline obtained by step 2), and derivant IPTG and arabinose is added
It is respectively 0.05~0.5mM and 0.002%~0.02% to final concentration, is then transferred to 28~30 DEG C, under the conditions of 180~220rpm
Continue culture 24~72 hours.
Preferably, the step 2) culture, condition of culture are：35 DEG C~37 DEG C, 180~220rpm.
In another embodiment, the side provided by the invention that isoprene is prepared using above-mentioned engineering colon bacillus
Method is that the engineering colon bacillus that will be built carries out activation acquisition seed liquor in suitable culture medium, by seed liquor with 1~2:
100~130 ratio accesses the suitable culture containing kanamycins and chloramphenicol and concentrates, 35 DEG C~37 DEG C, 180~220rpm items
It is cultivated to OD under part600Thalline is recycled when between 1.8~2, the thalline after recycling is resuspended with the above-mentioned culture mediums of 5mL and is added again
Enter in the saline bottle containing 95mL, meanwhile, be added derivant IPTG and arabinose to final concentration be respectively 0.05~0.5mM and
0.002%~0.02%, induction destination protein carries out overexpression.Then 28~30 DEG C are transferred to, under the conditions of 180~220rpm after
Continuous culture 24~72 hours；Timing takes culture medium upper air in culture bottle, passes through vapor detection product.
During fermenting and producing isoprene, the culture medium used in Recombinant organism includes selected by various be suitable for
The culture medium of host cell (Escherichia coli) growth, carbon source are preferably the methanol of low cost；Without using other carbon sources or have
Machine nitrogen source, other compositions are not to be particularly limited simply, such as inorganic nitrogen-sourced (ammonium chloride, ammonium sulfate may be selected in nitrogen source
Deng), preferably inexpensive is inorganic nitrogen-sourced.
What the present invention obtained has the beneficial effect that：
Recombinant organism constructed by the present invention has the spy that isoprene is synthesized using methanol as sole carbon source
Point, fermentation for 24 hours, can obtain the isoprene of 343mg/L, and the conversion ratio of methanol-isoprene can reach 21.83%, conversion
Rate is the 58.64% of theoretical yield.
Description of the drawings
Fig. 1 is the flow chart that isoprene approach is synthesized with methanol carbon source；
(in figure, solid line represents single step reaction, and dotted line represents multistep reaction).
Specific implementation mode
With reference to specific embodiment, the present invention will be further described, but the present invention should not be limited by the examples.
Material therefor, reagent, instrument and method in following embodiment are the routine in this field without specified otherwise
Material, reagent, instrument and method can be obtained by commercial channel.
Here, being illustrated to route of synthesis used in the present invention (Fig. 1).
Route of synthesis used in the present invention includes methanol metabolic pathway and isoprene route of synthesis.
Pentose phosphate pathway and glycolytic pathway mentioned below is the metabolic pathway having in itself in Bacillus coli cells.
Methanol metabolic pathway in the present invention, important reaction are：First is activated using methanol dehydrogenation ras GTPase activating protein ras-GTP ACT
The enzyme activity of alcohol dehydrogenase MDH；Methanol is oxidized to formaldehyde using methanol dehydrogenase MDH；Utilize 6- hexulose phosphate synzyme
RmpA, the X 5P reaction generated by formaldehyde and pentose phosphate pathway generate 6- hexulose phosphates；Using 6- phosphoric acid oneself
Ketose isomerase RmpB will generate 6- phosphoric acid fructofuranoses by 6- hexulose phosphates.
The fructose-1, 6-diphosphate kinases Pfk and fructose-diphosphonic acid aldehyde that 6- phosphoric acid fructofuranose is had by Escherichia coli itself contract
Enzyme Fba is converted into glyceraldehyde 3-phosphate, and glyceraldehyde 3-phosphate generates pyruvic acid after glycolytic pathway acts on.
Heretofore described has isoprene approach, refers to being overexpressed deoxy-D-xylulose sugar 5- phosphate synthase genes
Dxs, deoxy-D-xylulose sugar 5- phosphoric acid reductions enzyme dxr, Isopentenyl diphosphate isomerase idi and isoprenoid synthase gene
Isps is overexpressed the building process of the recombinant plasmid of said gene referring to patent CN102559769A.
The acquisition of 1 gene of embodiment
In the present embodiment, the formate dehydrogenase gene MDH (gene accession numbers from Methylotrophic bacillus are obtained
GI：662720579) gene order；6- hexulose phosphate synthase gene RmpA (gene accession number GI：40074227) base
Because of sequence；6- phosphoric acid -3- ketohexose isomerase gene RmpB (gene accession number GI：40074228) gene order；Methanol is de-
Hydrogen ras GTPase activating protein ras-GTP Gene A CT (gene accession number GI：22654851) gene order.Original sequence of the above gene is carried out
It is synthesized by the Suzhou bio tech ltd Jin Weizhi after codon optimization.The sequence of formate dehydrogenase gene MDH is such as after optimization
Shown in SEQ ID NO.1；The sequence of 6- hexulose phosphates synthase gene RmpA is as shown in SEQ ID NO.2 after optimization；Optimization
The sequence of 6- phosphoric acid -3- ketohexose isomerase genes RmpB is as shown in SEQ ID NO.3 afterwards；Methanol dehydrogenation enzyme activition egg after optimization
The sequence of white Gene A CT is as shown in SEQ ID NO.4.
The preparation of 2 recombinant plasmid of embodiment
By carrying out enzyme to the formate dehydrogenase gene MDH and plasmid pBAD18 obtained in embodiment 1 with EcoRI and SacI
It cuts, target fragment is recycled with plastic recovery kit, after digestion products recycling, by carrier：Target fragment is 1 in molar ratio:1 ratio
Example mixing, is added T4It is connected 6 hours at 16 DEG C after DNA ligase, obtains recombinant plasmid pBAD18-1；
By with SacI and KpnI to the 6- hexulose phosphate synthase gene RmpA and plasmid that are obtained in embodiment 1
PBAD18-1 carries out digestion, target fragment is recycled with plastic recovery kit, after digestion products recycling, by carrier：Target fragment is pressed
Molar ratio is 1:1 ratio mixing, connects 6 hours at 16 DEG C after T4DNA ligases are added, obtains recombinant plasmid pBAD18-
2；
By with SalI and SphI to the 6- phosphoric acid -3- ketohexose isomerase gene RmpB and plasmid that are obtained in embodiment 1
PBAD18-2 carries out digestion, target fragment is recycled with plastic recovery kit, after digestion products recycling, by carrier：Target fragment is pressed
Molar ratio is 1:1 ratio mixing, is added T4It is connected 6 hours at 16 DEG C after DNA ligase, obtains recombinant plasmid pBAD18-
3；
By with SphI to the methanol dehydrogenation ras GTPase activating protein ras-GTP Gene A CT that is obtained in embodiment 1 and plasmid pBAD18-3 into
Row digestion recycles target fragment, after digestion products recycling, by carrier with plastic recovery kit：Target fragment is 1 in molar ratio:1
Ratio mixing, connect 6 hours at 16 DEG C after T4DNA ligases are added, acquisition recombinant plasmid pBAD18-MDH-RmpA-
RmpB-ACT。
Plasmid pBAD18-3 and isoprene synthetic plasmid are imported E. coli JM109 (DE3) by embodiment 3
Synthesize isoprene
The recombinant plasmid pBAD18-3 of 2 gained of embodiment is added to E.coli simultaneously with isoprene synthetic plasmid
In JM109 (DE3) competent cell；Ice bath 20min, the competent cell after ice bath are put into 42 DEG C of water-bath heat shock 45s, heat shock
Ice bath 2min immediately afterwards；In super-clean bench, the centrifuge tube where the LB liquid medium to competent cell of 600 μ l sterilizings is added
It is interior；Centrifuge tube is placed in 37 DEG C of shaking table culture 1h；After shaking table culture, 500rpm centrifuges 2min, and supernatant is sucked out with liquid-transfering gun, stays
Cell is resuspended in a little supernatant, and is coated on the LB solid plates containing kanamycins and chloramphenicol；Tablet after coating is placed in
37 DEG C of constant incubators continue culture to growing monoclonal.
The engineered strain monoclonal of acquisition is added in the appropriate media containing kanamycins and chloramphenicol, at 37 DEG C,
Activation is carried out under the conditions of 180rpm and obtains seed liquor, by seed liquor with 1:The 100 above culture of ratio access, which is concentrated, to be continued to cultivate.
Cell concentration grows to OD600It is 2 or so, recycles thalline in an aseptic environment.Thalline after recycling carries out weight with 5mL culture mediums
It is outstanding, it is added in the saline bottle of the 500mL volumes containing 95mL culture mediums, while it is dense to end that derivant IPTG and arabinose is added
Degree is respectively 0.05mM and 0.02%, and is sealed using butyl rubber bung.30 DEG C are transferred to, continues to cultivate under the conditions of 180rpm, lure
It leads destination protein and carries out overexpression.After culture 24 hours, culture medium upper air in culture bottle is taken, is produced by vapor detection
Object.
After testing, the yield of isoprene is 217mg/L, and methanol to isoprene conversion ratio is 9.4%.
Plasmid pBAD18-3 and isoprene synthetic plasmid are imported E. coli BL21 (DE3) and closed by embodiment 4
At isoprene
The recombinant plasmid pBAD18-3 of 2 gained of embodiment is added to E.coli simultaneously with isoprene synthetic plasmid
In BL21 (DE3) competent cell；Ice bath 20min, the competent cell after ice bath are put into 42 DEG C of water-bath heat shock 45s, heat shock
Ice bath 2min immediately afterwards；In super-clean bench, the centrifuge tube where the LB liquid medium to competent cell of 600 μ l sterilizings is added
It is interior；Centrifuge tube is placed in 37 DEG C of shaking table culture 1h；After shaking table culture, 500rpm centrifuges 2min, and supernatant is sucked out with liquid-transfering gun, stays
Cell is resuspended in a little supernatant, and is coated on the LB solid plates containing kanamycins and chloramphenicol；Tablet after coating is placed in
37 DEG C of constant incubators continue culture to growing monoclonal.
The engineered strain monoclonal of acquisition is added in the appropriate media containing kanamycins and chloramphenicol, at 37 DEG C,
Activation is carried out under the conditions of 180rpm and obtains seed liquor, by seed liquor with 1:The 100 above culture of ratio access, which is concentrated, to be continued to cultivate.
Cell concentration grows to 0D600It is 2 or so, recycles thalline in an aseptic environment.Thalline after recycling carries out weight with 5mL culture mediums
It is outstanding, it is added in the saline bottle of the 500mL volumes containing 95mL culture mediums, while it is dense to end that derivant IPTG and arabinose is added
Degree is respectively 0.05mM and 0.02%, and is sealed using butyl rubber bung.30 DEG C are transferred to, continues to cultivate under the conditions of 180rpm, lure
It leads destination protein and carries out overexpression.After culture 24 hours, culture medium upper air in culture bottle is taken, is produced by vapor detection
Object.
After testing, the yield of isoprene is 297mg/L, and methanol to isoprene conversion ratio is 12.9%, relative to reality
The engineering bacteria in example 3 is applied, 36.8% and 37.2% has been respectively increased in yield and conversion ratio.
Plasmid pBAD18-MDH-RmpA-RmpB-ACT and isoprene synthetic plasmid are imported Escherichia coli by embodiment 5
E.coliBL21 (DE3) synthesizes isoprene
Simultaneously with isoprene synthetic plasmid by the recombinant plasmid pBAD18-MDH-RmpA-RmpB-ACT of 2 gained of embodiment
It is added in E.coli BL21 (DE3) competent cell；Ice bath 20min, the competent cell after ice bath are put into 42 DEG C of water-baths
Heat shock 45s, ice bath 2min immediately after heat shock；In super-clean bench, the LB liquid mediums of 600 μ l sterilizings is added to competent cell
In the centrifuge tube at place；Centrifuge tube is placed in 37 DEG C of shaking table culture 1h；After shaking table culture, 500rpm centrifuges 2min, uses liquid-transfering gun
Supernatant is sucked out, stays a little supernatant that cell is resuspended, and be coated on the LB solid plates containing kanamycins and chloramphenicol；Coating
Tablet afterwards is placed in 37 DEG C of constant incubators, continues culture to growing monoclonal.
The engineered strain monoclonal of acquisition is added in the appropriate media containing kanamycins and chloramphenicol, at 37 DEG C,
Activation is carried out under the conditions of 180rpm and obtains seed liquor, by seed liquor with 1:The 100 above culture of ratio access, which is concentrated, to be continued to cultivate.
Cell concentration grows to 0D600It is 2 or so, recycles thalline in an aseptic environment.Thalline after recycling carries out weight with 5mL culture mediums
It is outstanding, it is added in the saline bottle of the 500mL volumes containing 95mL culture mediums, while it is dense to end that derivant IPTG and arabinose is added
Degree is respectively 0.05mM and 0.02%, and is sealed using butyl rubber bung.30 DEG C are transferred to, continues to cultivate under the conditions of 180rpm, lure
It leads destination protein and carries out overexpression.After culture 24 hours, culture medium upper air in culture bottle is taken, is produced by vapor detection
Object.
After testing, the yield of isoprene is 343mg/L, and methanol to isoprene conversion ratio is 21.83%.Relative to reality
Do not have to be overexpressed the engineering bacteria of methanol dehydrogenation ras GTPase activating protein ras-GTP Gene A CT in applying 4 in example, yield and conversion ratio are respectively increased
32.32% and 69.22%.
The above embodiments are merely illustrative of the technical solutions of the present invention, rather than is limited；Although with reference to above-mentioned reality
Example is applied to explain the present invention in detail, it for those of ordinary skill in the art, still can be to aforementioned implementation
Technical solution recorded in example is modified or equivalent replacement of some of the technical features；And these are changed or replace
It changes, the spirit and scope for claimed technical solution of the invention that it does not separate the essence of the corresponding technical solution.
Claims (7)
1. a kind of catalysis methanol prepares the Recombinant organism of isoprene, which is characterized in that have isoprene to close
At approach, meanwhile, it is overexpressed formate dehydrogenase gene MDH, 6- hexulose phosphate synthase gene RmpA, 6- phosphoric acid -3- hexanones
Sugared isomerase gene RmpB and methanol dehydrogenation ras GTPase activating protein ras-GTP Gene A CT, the formate dehydrogenase gene MDH derive from methyl
Auxotype bacillus (Bacillus methanolicus), gene accession number GI：662720579；The 6- hexulose phosphates
Synthase gene RmpA derives from Methylotrophic bacillus, gene accession number GI：40074227；6- phosphoric acid-the 3- oneself
Ketose isomerase gene RmpB derives from Methylotrophic bacillus, gene accession number GI：40074228；The methanol is de-
Hydrogen ras GTPase activating protein ras-GTP act gene derives from Methylotrophic bacillus, gene accession number GI：AY128667.1；It is described to have
Isoprene route of synthesis refers to being overexpressed deoxy-D-xylulose sugar 5- phosphate synthase gene dxs, deoxy-D-xylulose sugar 5- phosphoric acid reductions
Enzyme dxr, Isopentenyl diphosphate isomerase idi and isoprenoid synthase gene Isps.
2. the construction method of Recombinant organism described in a kind of claim 1, which is characterized in that steps are as follows：
1) clone or synthesize obtain formate dehydrogenase gene MDH, 6- hexulose phosphate synthase gene RmpA, 6- phosphoric acid -3- oneself
Ketose isomerase gene RmpB and methanol dehydrogenation ras GTPase activating protein ras-GTP act gene, and gained gene and plasmid pBAD18 digestions are returned
It receives, target fragment and carrier；
2) it is connected using ligase after mixing the carrier obtained by step 1) according to equimolar ratio with target fragment, and is transferred to impression
In state cell, screening positive clone；
3) incubation step 2) obtained by positive colony, and extract plasmid, four genes described in step 1) be connected to plasmid
On pBAD18, recombinant plasmid pBAD18-MDH-RmpA-RmpB-ACT is obtained；
4) recombinant plasmid of the structure with isoprene route of synthesis, and by the recombination obtained by the recombinant plasmid of gained and step 3)
Plasmid pBAD18-MDH-RmpA-RmpB-ACT is transformed into together in E. coli BL21 (DE3), obtains large intestine bar
Bacterium genetic engineering bacterium.
3. method according to claim 2, which is characterized in that the step 2) ligase is T4DNA ligase, Connection Time
It is 6~12 hours；The competent cell, to pass through heat-shock transformed E.coliDH5 α competent cells；The screening is profit
It is incubated overnight with coating chloramphenicol or kalamycin resistance tablet, PCR screenings.
4. method according to claim 2, which is characterized in that step 4) the recombination matter with isoprene route of synthesis
Grain, including deoxy-D-xylulose sugar 5- phosphate synthase gene dxs, deoxy-D-xylulose sugar 5- phosphoric acid reduction enzyme dxr, isopentenyl pyrophosphate
Isomerase idi and isoprenoid synthase gene Isps.
5. application of the Recombinant organism described in claim 1 in producing isoprene.
6. being applied described in claim 5, which is characterized in that steps are as follows：
1) Recombinant organism is activated, seed liquor is obtained；
2) by the seed liquor obtained by step 1), and the culture medium containing kanamycins and chloramphenicol, according to seed liquor：Culture medium=
1~2:It is cultivated to OD after 100~130 ratio inoculation6001.8~2, centrifugation obtains thalline；
3) it is added in fresh culture after being resuspended to the thalline obtained by step 2), it is dense to end that derivant IPTG and arabinose is added
Degree is respectively 0.05~0.5mM and 0.002%~0.02%, is then transferred to 28~30 DEG C, continues to train under the conditions of 180~220rpm
It supports 24~72 hours.
7. applying according to claim 6, which is characterized in that the step 2) culture, condition of culture are：35 DEG C~37 DEG C,
180~220rpm.
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| CN102559769A (en) * | 2010-12-20 | 2012-07-11 | 中国科学院青岛生物能源与过程研究所 | Reconstituted cell for isoprene and preparation method of reconstituted cell |
| CN104789512A (en) * | 2014-01-16 | 2015-07-22 | 清华大学 | Isoprene producing bacterial and isoprene producing method |
| CN104995293A (en) * | 2012-12-17 | 2015-10-21 | 基因组股份公司 | Microorganisms and methods for enhancing the availability of reducing equipments in the presence of methanol, and for producing adipate, 6-aminocaproate, hexamethylenediamine or caprolactam related thereto |
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| CN102559769A (en) * | 2010-12-20 | 2012-07-11 | 中国科学院青岛生物能源与过程研究所 | Reconstituted cell for isoprene and preparation method of reconstituted cell |
| CN104995293A (en) * | 2012-12-17 | 2015-10-21 | 基因组股份公司 | Microorganisms and methods for enhancing the availability of reducing equipments in the presence of methanol, and for producing adipate, 6-aminocaproate, hexamethylenediamine or caprolactam related thereto |
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